Bone Matrix and Bone Resorption

描述（由申请人提供）：这些研究都来源于我们之前探索骨基质与骨吸收相互作用的工作，重点关注影响骨骼发育和骨吸收的基因和其他因素。Specific Aim 1提出绘制两个独特的突变，负责大鼠破骨细胞发育和功能的失败。这一目标将采用经典遗传学结合分子定位和定位克隆技术来确定导致门牙缺失（ia）和无牙（tl）大鼠突变的骨质疏松表型的基因。这些我们维持多年的自交系将与一个高度多态的菌株进行异交。一系列PCR反应、连锁研究和最终详细的序列分析将用于鉴定突变基因。目前正在评估来自tl大鼠新发现区域的候选细胞。特异性目的2探讨成骨细胞分泌结缔组织生长因子（CTGF）与破骨细胞募集和活化之间的功能关系。我们已经在三种大鼠和一种小鼠的成骨细胞中发现了CTGF mRNA和蛋白质在体内的上调，所有这些突变都在不同的点上阻断了破骨细胞的分化和/或功能。CTGF过表达的共性表明，将CTGF降至正常水平需要骨吸收。我们将对这一假设进行验证：(1)通过治疗突变动物并跟踪体内对CTGF水平的影响；(2)利用破骨细胞和骨器官培养系统，通过抗CTGF抗体和用重组CTGF使破骨细胞α (v) β(3)整合素饱和来阻止CTGF/破骨细胞结合，并测量对再吸收的影响；(3)再吸收培养上清检测其下调成骨细胞CTGF表达的能力。特异性目的3将确定胶原基因转换失调是否是颅面联合软骨和缝合线生长异常的共同特征。我们已经在大鼠的两个关键生长中心发现了胶原基因转换（I型和II型以及II型和X型）的失败。我们将把这些研究扩展到颅底的联合软骨，并使用这种方法筛选一系列颅面和/或骨质疏松突变的小鼠，以寻找类似的失败。这将确定伴随颅面缺陷的胶原表达异常的频率，是否构成颅面生长缺陷的潜在“读出”，因此是否有必要进行机械的未来研究。假设/具体目标对骨质疏松突变中异常基因表达的深入研究将揭示更多参与调节正常骨骼代谢的基因。我们以前已经使用mRNA的差异显示来发现几个重要的骨骼基因。我们将使用高密度基因阵列技术来扩展这些研究，以比较正常、骨质疏松和治愈的骨质疏松骨RNA，以确定更多在骨质疏松突变中上调或下调的基因和途径，并以此作为体内破骨细胞分化的基础。这种方法也将允许我们跟踪异常表达的持久性或在细胞因子治疗恢复吸收后恢复正常。

DESCRIPTION (provided by applicant): These studies, all derived from our previous work exploring the interactions of bone matrix and bone resorption, focus on genes and other factors impacting skeletal development and resorption. Specific Aim 1 proposes mapping two unique mutations responsible for the failure of osteoclast development and function in the rat. This aim will be pursued using classical genetics combined with molecular mapping and positional cloning techniques to identify the genes responsible for the osteopetrotic phenotypes of the incisor absent (ia) and toothless (tl) rat mutations. These inbred strains that we have maintained for many years will be outcrossed with a highly polymorphic strain. A series of PCR reactions, linkage studies, and ultimately detailed sequence analyses will be used to identify the mutated genes. Candidates from a newly identified region in the tl rat are currently being evaluated. Specific Aim 2 explores the functional relationship between connective tissue growth factor (CTGF) secretion by osteoblasts and osteoclast recruitment and activation. We have shown up-regulation of CTGF mRNA and protein in vivo by osteoblasts in three osteopetrotic mutations in the rat and one in the mouse, all of which intercept osteoclast differentiation and/or function at distinct points. The commonality of CTGF over-expression implies that bone resorption is required to reduce CTGF to normal levels. We will test this hypothesis: (1) by curing mutant animals and following the impact on CTGF levels in vivo; (2) using osteoclast and bone organ culture systems, CTGF/osteoclast binding will be prevented by anti-CTGF antibodies and by saturating osteoclast alpha(v)beta(3) integrins with recombinant CTGF, and the effect on resorption will measured; (3) supernatants from resorbing cultures will be tested for their ability to down-regulate CTGF expression by osteoblasts. Specific Aim 3 will establish whether collagen gene switching dis-regulation is a common feature of growth abnormalities at craniofacial synchondroses and sutures. We have shown failures of collagen gene switching (types I and Ill and types II and X) at two key growth centers in the tl rat. We will extend these studies to the synchondroses of the skull base, and use this approach to screen a series of mice with craniofacial and/or osteopetrotic mutations for similar failures. This will establish how frequently collagen expression abnormalities accompany craniofacial defects, whether it constitutes a potential "read-out" for craniofacial growth defects, and therefore whether mechanistic future investigations are warranted. Hypothesis/Specific Aim 4. Extending studies of abnormal gene expression in osteopetrotic mutations will uncover more genes involved in regulating normal skeletal metabolism. We have previously used differential display of mRNA to discover several important skeletal genes. We will extend these studies using high-density gene array technology to compare normal, osteopetrotic, and cured osteopetrotic bone RNA to identify more genes and pathways that are up- or down-regulated in osteopetrotic mutations and that underlie osteoclast differentiation in vivo. This method will also permit us to follow the persistence of abnormal expression or its return to normal following restoration of resorption by cytokine treatments.